cDNA prepared from the single-stranded circular RNA genome of hepatitis delta virus was cloned in lambda gt11 by using RNA from the liver of an infected woodchuck. From the sequence of overlapping clones, we assembled the full sequence of 1,679 nucleotides. The sequence indicated an exceptional ability for intramolecular base pairing, yielding a rod structure with at least 70% of the bases paired and a predicted free energy of -805 kcal (-3,368 kJ)/mol. Three of the lambda clones contained sequences that were not only expressed as fusion proteins with beta-galactosidase but were recognized by human hepatitis delta virus-specific antibody. These clones were sequenced so as to establish the reading frame of the delta antigen on the antigenomic strand. The fusion protein produced by one clone was purified by immunoaffinity chromatography and then was used to raise rabbit antibodies specific for the delta antigen.